Skip to main content

Advertisement

SpringerLink
Log in

In vitro testing for anti-inflammatory properties of compounds employing peripheral blood mononuclear cells freshly isolated from healthy donors

  • Original Research Paper
  • Published:
Inflammation Research Aims and scope Submit manuscript

Abstract

Introduction

Inflammation is crucially involved in a variety of diseases like autoimmune syndromes, cardiovascular and neurodegenerative disorders, cancer, sepsis and allograft rejection.

Methods

Freshly isolated human peripheral blood mononuclear cells (PBMCs) are used as a screening assay for anti-inflammatory properties of compounds. Determinations of neopterin production by ELISA and of tryptophan degradation by HPLC are used as read-outs. Results are compared with further markers of immune response and oxidative stress.

Results

Phytohaemagglutinin induced significant tryptophan degradation and neopterin formation in PBMC, which correlated with IFN-γ, TNF-α, soluble cytokine receptors and isoprostane-8. Addition of vitamin C and E suppressed the responses dose-dependently.

Discussion

The determination of tryptophan degradation and neopterin production in PBMC reflects various pro- and anti-inflammatory cascades that are of relevance also in patients. It constitutes a robust and reliable approach to screen anti-inflammatory or immunosuppressive drugs and may improve throughput, speed and cost-effectiveness in drug discovery.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Karagiannidis C, Rückert B, Hense G, Willer G, Menz G, Blaser K, Schmidt-Weber CB. Distinct leukocyte redistribution after glucocorticoid treatment among difficult-to-treat asthmatic patients. Scand J Immunol. 2005;61:187–96.

    Article  CAS  PubMed  Google Scholar 

  2. Bock HA. Steroid-resistant kidney transplant rejection: diagnosis and treatment. J Am Soc Nephrol. 2001;12:S48–52.

    PubMed  Google Scholar 

  3. Romagnani S. Immunologic influences on allergy and the TH1/TH2 balance. J Allergy Clin Immunol. 2004;113:395–400.

    Article  CAS  PubMed  Google Scholar 

  4. Nathan CF, Murray HW, Wiebe ME, Rubin BY. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med. 1983;158:670–89.

    Article  CAS  PubMed  Google Scholar 

  5. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1. EMBO J. 1991;10:2247–58.

    CAS  PubMed  Google Scholar 

  6. Asehnoune K, Strassheim D, Mitra S, Kim JY, Abraham E. Involvement of reactive oxygen species in toll-like receptor 4-dependent activation of NF-kappaB. J Immunol. 2004;15:2522–9.

    Google Scholar 

  7. Aggarwal BB. Nuclear factor-κB: the enemy within. Cancer Cell. 2004;6:203–8.

    Article  CAS  PubMed  Google Scholar 

  8. Min KJ, Jou I, Joe E. Plasminogen-induced IL-1beta and TNF-alpha production in microglia is regulated by reactive oxygen species. Biochem Biophys Res Comm. 2003;312:969–74.

    Article  CAS  PubMed  Google Scholar 

  9. Fuchs D, Hausen A, Reibnegger G, Werner ER, Dierich MP, Wachter H. Neopterin as a marker for activated cell-mediated immunity: application in HIV infection. Immunol Today. 1988;9:150–5.

    Article  CAS  PubMed  Google Scholar 

  10. Murr C, Widner B, Wirleitner B, Fuchs D. Neopterin as a marker for immune system activation. Curr Drug Metab. 2002;3:175–87.

    Article  CAS  PubMed  Google Scholar 

  11. Murr C, Fuith LC, Widner B, Wirleitner B, Baier-Bitterlich G, Fuchs D. Increased neopterin concentrations in patients with cancer: indicator of oxidative stress? Anticancer Res. 1999;19:1721–8.

    CAS  PubMed  Google Scholar 

  12. Hamerlinck FF. Neopterin: a review. Exp Dermatol. 1999;8:167–76.

    Article  CAS  PubMed  Google Scholar 

  13. Grammer TB, Fuchs D, Böhm BO, Winkelmann BR, Maerz W. Neopterin as a predictor of total and cardiovascular mortality in individuals undergoing angiography in the Ludwigshafen Risk and Cardiovascular Health study. Clin Chem. 2009;55:115–46.

    Article  Google Scholar 

  14. Byrne GI, Lehmann LK, Landry GJ. Induction of tryptophan catabolism is the mechanism for gamma-interferon-mediated inhibition of intracellular Chlamydia psittaci replication in T24 cells. Infect Immun. 1986;53:347–51.

    CAS  PubMed  Google Scholar 

  15. Fuchs D, Möller AA, Reibnegger G, Stöckle E, Werner ER, Wachter H. Decreased serum tryptophan in patients with HIV-1 infection correlates with increased serum neopterin and with neurologic/psychiatric symptoms. J Acquir Immune Defic Syndr. 1990;3:873–6.

    CAS  PubMed  Google Scholar 

  16. Schroecksnadel K, Wirleitner B, Winkler C, Fuchs D. Monitoring tryptophan metabolism in chronic immune activation. Clin Chim Acta. 2006;364:82–90.

    Article  CAS  Google Scholar 

  17. Huengsberg M, Winer JB, Gompels M, Round R, Ross J, Shahmanesh M. Serum kynurenine-to-tryptophan ratio increases with progressive disease in HIV-infected patients. Clin Chem. 1998;44:858–62.

    CAS  PubMed  Google Scholar 

  18. Weinlich G, Murr C, Richardsen L, Winkler C, Fuchs D. Decreased serum tryptophan concentration predicts poor prognosis in malignant melanoma patients. Dermatology. 2007;214:8–14.

    Article  PubMed  Google Scholar 

  19. Ploder M, Spittler A, Schroecksnadel K, Neurauter G, Pelinka LM, Roth E, et al. Tryptophan degradation in multiple trauma patients: survivors versus non-survivors. Clin Sci. 2009;116:593–8.

    Article  CAS  PubMed  Google Scholar 

  20. Chen Y, Guillemin GJ. Kynurenine pathway metabolites in humans: disease and healthy states. Int J Tryptophan Res. 2009;2:1–19.

    Google Scholar 

  21. Tattevin P, Monnier D, Tribut O, Dulong J, Bescher N, Mourcin F, et al. Enhanced indoleamine 2,3-dioxygenase activity in patients with severe sepsis and septic shock. J Infect Dis. 2010;201:956–66.

    Article  CAS  PubMed  Google Scholar 

  22. Weiss G, Murr C, Zoller H, Haun M, Widner B, Ludescher C, et al. Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells. Clin Exp Immunol. 1999;116:435–40.

    Article  CAS  PubMed  Google Scholar 

  23. Winkler C, Ueberall F, Fuchs D. In vitro testing for anti-inflammatory properties of compounds. Clin Chem. 2006;52:1201–2.

    Article  CAS  PubMed  Google Scholar 

  24. Schroecksnadel K, Fischer B, Schennach H, Weiss G, Fuchs D. Antioxidants down-regulate Th1-type immune response in vitro. Drug Metab Lett. 2007;1:166–71.

    Article  CAS  PubMed  Google Scholar 

  25. Billiau AD, Roskams T, Van Damme-Lombaerts R, Matthys P, Wouters C. Macrophage activation syndrome: characteristic findings on liver biopsy illustrating the key role of activated, IFN-gamma-producing lymphocytes and IL-6- and TNF-alpha-producing macrophages. Blood. 2005;105:1648–51.

    Article  CAS  PubMed  Google Scholar 

  26. Murray HW. Interferon-gamma, the activated macrophage, and host defense against microbial challenge. Ann Intern Med. 1988;108:595–608.

    CAS  PubMed  Google Scholar 

  27. Diez-Ruiz A, Tilz GP, Zangerle R, Baier-Bitterlich G, Wachter H, et al. Soluble receptors for tumor necrosis factor in clinical laboratory diagnosis. Eur J Haematol. 1995;54:1–8.

    Article  CAS  PubMed  Google Scholar 

  28. Waldmann TA. The IL-2/IL-15 receptor systems: targets for immunotherapy. J Clin Immunol. 2002;22:51–6.

    Article  CAS  PubMed  Google Scholar 

  29. Sperner-Unterweger B, Neurauter G, Klieber M, Kurz K, Meraner V, Zeimet A, et al. Enhanced tryptophan degradation in patients with ovarian carcinoma correlates with several serum soluble immune activation markers. Immunobiology (in press).

  30. Ploder M, Spittler A, Schroecksnadel K, Neurauter G, Pelinka LM, Roth E, et al. Accelerated tryptophan degradation in trauma and sepsis patients is related to pro-inflammatory response and to the diminished in vitro response of monocytes. Pteridines. 2009;19:54–61.

    Google Scholar 

  31. Montuschi P, Barnes P, Roberts LJ 2nd. Insights into oxidative stress: the isoprostanes. Curr Med Chem. 2007;14:703–17.

    Article  CAS  PubMed  Google Scholar 

  32. Winkler C, Schroecksnadel K, Schennach H, Fuchs D. Vitamin C and E suppress mitogen-stimulated peripheral blood mononuclear cells in vitro. Int Arch Allergy Immunol. 2007;142:127–32.

    Article  CAS  PubMed  Google Scholar 

  33. Neurauter G, Wirleitner B, Laich A, Schennach H, Weiss G, Fuchs D. Atorvastatin suppresses interferon-γ-induced neopterin formation and tryptophan degradation in human peripheral blood mononuclear cells and in monocytic cell lines. Clin Exp Immunol. 2003;131:264–7.

    Article  CAS  PubMed  Google Scholar 

  34. Widner B, Werner ER, Schennach H, Wachter H, Fuchs D. Simultaneous measurement of serum tryptophan and kynurenine by HPLC. Clin Chem. 1997;43:2424–6.

    CAS  PubMed  Google Scholar 

  35. Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, Reibnegger G, et al. Immune response-associated production of neopterin. Release from macrophages primarily under control of interferon-gamma. J Exp Med. 1984;160:310–6.

    Article  CAS  PubMed  Google Scholar 

  36. Wirleitner B, Reider D, Ebner S, Böck G, Widner B, Jaeger M, et al. Monocyte-derived dendritic cells release neopterin. J Leukoc Biol. 2002;72:1148–53.

    CAS  PubMed  Google Scholar 

  37. Werner ER, Werner-Felmayer G, Fuchs D, Hausen A, Reibnegger G, Wachter H. Parallel induction of tetrahydrobiopterin biosynthesis and indoleamine 2,3-dioxygenase activity in human cells and cell lines by interferon-gamma. Biochem J. 1989;262:861–6.

    CAS  PubMed  Google Scholar 

  38. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, et al. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med. 2003;9:1269–74.

    Article  CAS  PubMed  Google Scholar 

  39. Samsonov MY, Tilz GP, Egorova O, Reibnegger G, Balabanova RM, Nassonov EL, et al. Serum soluble markers of immune activation and disease activity in systemic lupus erythematosus. Lupus. 1995;4:29–32.

    Article  CAS  PubMed  Google Scholar 

  40. Diez-Ruiz A, Tilz GP, Gutierrez-Gea F, Gil-Extremera B, Murr C, Wachter H, et al. Neopterin and soluble tumor necrosis factor receptor type I in alcohol-induced cirrhosis. Hepatology. 1985;21:976–8.

    Article  Google Scholar 

  41. Tan PH, Sagoo P, Chan C, Yates JB, Campbell J, Beutelspacher SC, et al. Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells. J Immunol. 2005;174:7633–44.

    CAS  PubMed  Google Scholar 

  42. Wirleitner B, Schroecksnadel K, Winkler C, Schennach H, Fuchs D. Resveratrol suppresses interferon-γ-induced biochemical pathways in human peripheral blood mononuclear cells in vitro. Immunol Lett. 2005;100:159–63.

    Article  CAS  PubMed  Google Scholar 

  43. Zvetkova E, Wirleitner B, Tram NT, Schennach H, Fuchs D. Aqueous extracts of Crinum latifolium (L.) and Camellia sinensis show immunomodulatory properties in human peripheral blood mononuclear cells. Int Immunopharmacol. 2001;1:2143–50.

    Article  CAS  PubMed  Google Scholar 

  44. Neurauter G, Wirleitner B, Schroecksnadel K, Schennach H, Fuchs D. Wine and grape juice modulate interferon-γ-induced neopterin production and tryptophan degradation in human PBMC. Pteridines. 2004;15:1–9.

    CAS  Google Scholar 

  45. Winkler C, Wirleitner B, Schroecksnadel K, Schennach H, Fuchs D. Beer down-regulates activated peripheral blood mononuclear cells in vitro. Int Immunopharmacol. 2006;6:390–5.

    Article  CAS  PubMed  Google Scholar 

  46. Schroecksnadel S, Sucher M, Schroecksnadel K, Sucher R, Brandacher G, Margreiter R, Fuchs D. Influence of immunosuppressive agents on neopterin production and tryptophan degradation in human peripheral blood mononuclear cells. Pteridines. 2009;20:34–5.

    Google Scholar 

  47. Schroecksnadel K, Winkler C, Wirleitner B, Schennach H, Fuchs D. Aspirin down-regulates tryptophan degradation in stimulated human peripheral blood mononuclear cells in vitro. Clin Exp Immunol. 2005;140:41–5.

    Article  CAS  PubMed  Google Scholar 

  48. Jenny M, Santer E, Pirich E, Schennach H, Fuchs D. Δ9-Tetrahydrocannabinol and cannabidiol modulate mitogen-induced tryptophan degradation and neopterin formation in peripheral blood mononuclear cells in vitro. J Neuroimmunol. 2009;207:75–82.

    Article  CAS  PubMed  Google Scholar 

  49. Girgin G, Baydar T, Ledochowski M, Schennach H, Bolukbasi DN, Sorkun K, Salih B, Sahin G, Fuchs D. Immunomodulatory effects of Turkish propolis: changes in neopterin release and tryptophan degradation. Immunobiology. 2009;214:129–34.

    Article  CAS  PubMed  Google Scholar 

  50. Winkler C, Wirleitner B, Schroecksnadel K, Schennach H, Mur E, Fuchs D. In vitro effects of two extracts and two pure alkaloid preparations of Uncaria tomentosa on peripheral blood mononuclear cells. Planta Med. 2004;70:205.

    Article  CAS  PubMed  Google Scholar 

  51. Winkler C, Wirleitner B, Schroecksnadel K, Schennach H, Fuchs D. St. John’s wort (Hypericum perforatum) counteracts cytokine-induced tryptophan catabolism in vitro. Biol Chem. 2004;385:1197–202.

    Article  CAS  PubMed  Google Scholar 

  52. Jenny M, Santer E, Klein A, Ledochowski M, Schennach H, Ueberall F, et al. Cacao extracts suppress tryptophan degradation of mitogen-stimulated peripheral blood mononuclear cells. J Ethnopharmacol. 2009;207:75–82.

    CAS  Google Scholar 

  53. Winkler C, Frick B, Schroecksnadel K, Schennach H, Fuchs D. Food preservatives sodium sulfite and sorbic acid suppress mitogen- stimulated peripheral blood mononuclear cells. Food Chem Toxicol. 2006;44:2003–7.

    Article  CAS  PubMed  Google Scholar 

  54. Winkler C, Wirleitner B, Schroecksnadel K, Schennach H, Fuchs D. In vitro effects of beet root juice on stimulated and unstimulated peripheral blood mononuclear cells. Am J Biochem Biotechnol. 2005;1:181–6.

    Google Scholar 

  55. Maier E, Kurz K, Jenny M, Schennach H, Ueberall F, Fuchs D. Food preservatives sodium benzoate and propionic acid and colorant curcumin suppress Th1-type immune response in vitro. Food Chem Toxicol. 2010;48(7):1950–6.

    Article  CAS  PubMed  Google Scholar 

  56. Aggarwal BB, Van Kuiken ME, Iyer LH, Harikumar KB, Sung B. Molecular targets of nutraceuticals derived from dietary spices: potential role in suppression of inflammation and tumorigenesis. Exp Biol Med. 2009;234:825–49.

    Article  CAS  Google Scholar 

  57. Nathan CF. Peroxide and pteridine: a hypothesis on the regulation of macrophage antimicrobial activity by interferon-γ. In: Gresser I, Vilcek J, editors. Interferon 7. London: Academic Press; 1986. p. 125–43.

    Google Scholar 

  58. Murr C, Schroecksnadel K, Winklhofer-Roob BM, Mangge H, Böhm BO, Winkelmann BR, et al. Inverse association between serum concentrations of neopterin and antioxidants in patients with and without angiographic coronary artery disease. Atherosclerosis. 2009;202:543–9.

    Article  CAS  PubMed  Google Scholar 

  59. Hronek M, Zadak Z, Solichova D, Jandik P, Melichar B. The association between specific nutritional antioxidants and manifestation of colorectal cancer. Nutrition. 2000;16:189–91.

    Article  CAS  PubMed  Google Scholar 

  60. Neurauter G, Grahmann AV, Klieber M, Zeimet A, Ledochowski M, Sperner-Unterweger B, et al. Serum phenylalanine concentrations in patients with ovarian carcinoma correlate with concentrations of immune activation markers and of isoprostane-8. Cancer Lett. 2008;272:141–7.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Miss Maria Gleinser for excellent technical assistance.

Author information

Authors and Affiliations

  1. Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz Pregl Strasse 3, 6020, Innsbruck, Austria

    M. Jenny, M. Klieber, S. Schroecksnadel & Dietmar Fuchs

  2. Department of Internal Medicine, Innsbruck Medical University, Innsbruck, Austria

    K. Kurz & M. Ledochowski

  3. Institute for Blood Transfusion and Immunology, University Clinics, Innsbruck, Austria

    H. Schennach

  4. Department of Pediatrics, University Hospital of Vienna, Vienna, Austria

    D. Zaknun

Authors
  1. M. Jenny
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Klieber
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. Zaknun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. Schroecksnadel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K. Kurz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. Ledochowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. H. Schennach
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dietmar Fuchs
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dietmar Fuchs.

Additional information

Responsible Editor: Liwu Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jenny, M., Klieber, M., Zaknun, D. et al. In vitro testing for anti-inflammatory properties of compounds employing peripheral blood mononuclear cells freshly isolated from healthy donors. Inflamm. Res. 60, 127–135 (2011). https://doi.org/10.1007/s00011-010-0244-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00011-010-0244-y

Keywords

Search

Navigation